Photodynamic therapy (PDT) of tumors begins with light activation of a photosensitizer in tumor cells, but mechanisms of cell death have remained elusive. The phthalocyanine photosensitizer Pc 4 interacts directly with the cardiolipin (CL) probe nonyl-acridine orange (NAO), as evidenced by fluorescence resonance energy transfer (FRET) between the two dyes. CL is a phospholipid found only in the inner mitochondrial membrane and at contact sites with the outer membrane. The contact sites also house components of the permeability transition pore, which interact with the anti-apoptotic proteins Bcl-2 and Bcl-xL, identified targets of Pc 4-PDT. The central hypothesis is: Upon photoirradiation, CL oxidation primes cytochrome c for release from mitochondria and contributes to the observed loss of Bcl-2 and Bcl-xL. The PDT-induced changes in the mitochondrial membranes, especially in CL and associated proteins, are sufficient for cell death. There are four Specific Aims. Aim 1 will define the role of Bcl-2 and Bcl-xL in determining the level of CL and NAO binding to it. Preliminary correlation of CL level, as defined by NAO fluorescence, with Bcl-2 overexpression will be expanded in a series of MCF-7-derived cell lines overexpressing Bcl-2, Bcl-2 mutants, or Bcl-xL. The goal of Aim 2 is to synthesize and test analogs of NAO with improved spectral properties for optimal detection of FRET with Pc 4 and other photosensitizers. Analogs will be synthesized by extension of the acridine ring system or by appending another fluorophore. They will be characterized for their photophysical properties, affinity for CL, and interaction with Pc 4. Aim 3 will characterize the oxidative changes in CL that result from PDT or other oxidizing agents. Cells or liposomes will be exposed to PDT or to non-PDT oxidizing agents. CL will be isolated and the amount and degree of oxidation will be measured by HPLC/MS analysis. Aim 4 will evaluate the relevance of CL sites to the PDT response of cells. Photodamage to Bcl-2 and Bcl-xL, loss of mitochondrial membrane potential, the release of cytochrome c, and cell death will be triggered by the transfer of energy from NAO or a superior analog to Pc 4. Responses will be compared to direct activation of Pc 4 by 672 nm laser light. The findings will elucidate the earliest photodynamic targets and events and determine their contribution to cellular responses to PDT. [unreadable] [unreadable]